Adler JH, Trainin D. The apparent effect of alfalfa on the reproductive performance of dairy
cattle. In: Proceedings of the Fourth International Congress on Animal Reproduction
and Artificial Insemination; June 5-9, 1961; The Hague, the Netherlands.

Fomon SJ, Rogers RR, Ziegler EE, Nelson SE, Thomas LN. Indices of fatness and serum cholesterol at age eight years in relation
to feeding and growth during early infancy. Pediatr Res.1984;18:1233-1238.

Author Affiliations: Center for Clinical Epidemiology and Biostatistics and Department of Biostatistics and Epidemiology (Drs Strom, Barnhart, Sammel, and Macones and Ms Schinnar), Division of General Internal Medicine, Department of Medicine (Dr Strom), Department of Obstetrics and Gynecology (Drs Barnhart and Macones), and Division of Gastroenterology and Nutrition, Children's Hospital, and Department of Pediatrics (Dr Stallings), University of Pennsylvania School of Medicine, Philadelphia; and Fomon Infant Nutrition Unit, Department of Pediatrics, University of Iowa, Iowa City (Dr Ziegler, Mr Nelson, and Mss Drulis and Hanson).

Context A large body of evidence documents the role of phytoestrogens in influencing
hormone-dependent states. Infants fed soy formula receive high levels of phytoestrogens,
in the form of soy isoflavones, during a stage of development at which permanent
effects are theoretically possible. However, a paucity of data exists on the
long-term effects of infant soy formulas.

Objective To examine the association between infant exposure to soy formula and
health in young adulthood, with an emphasis on reproductive health.

Design, Setting, and Participants Retrospective cohort study conducted from March to August 1999 among
adults aged 20 to 34 years who, as infants, participated during 1965-1978
in controlled feeding studies conducted at the University of Iowa, Iowa City
(248 were fed soy formula and 563 were fed cow milk formula during infancy).

Main Outcome Measures Self-reported pubertal maturation, menstrual and reproductive history,
height and usual weight, and current health, compared based on type of formula
exposure during infancy.

Results No statistically significant differences were observed between groups
in either women or men for more than 30 outcomes. However, women who had been
fed soy formula reported slightly longer duration of menstrual bleeding (adjusted
mean difference, 0.37 days; 95% confidence interval [CI], 0.06-0.68), with
no difference in severity of menstrual flow. They also reported greater discomfort
with menstruation (unadjusted relative risk for extreme discomfort vs no or
mild pain, 1.77; 95% CI, 1.04-3.00).

Conclusions Exposure to soy formula does not appear to lead to different general
health or reproductive outcomes than exposure to cow milk formula. Although
the few positive findings should be explored in future studies, our findings
are reassuring about the safety of infant soy formula.

Soy isoflavones, a subcategory of phytoestrogens, are naturally occurring
plant compounds. Phytoestrogens have been shown to bind to estrogen receptors
in the adult1- 3
and to act either as estrogens4 or as antiestrogens.5,6 For example, phytoestrogens can block
the action of endogenous estrogens on the uterus.5- 7

A large body of evidence documents the role of phytoestrogens in influencing
hormone-dependent states.4,8- 10
Dietary phytoestrogens during adulthood have been suggested in numerous epidemiological
studies to be protective against cancer of the prostate, colon, rectum, stomach,
breast, and lung and to exert similarly protective effects against chronic
conditions such as atherosclerosis and osteoporosis.11- 15
Evidence in some animal species of a contraceptive or sexual development effect
from dietary phytoestrogens16- 26
has led some to suggest that dietary habits should be investigated in women
with differences in menstrual cycle length.27
Lowered sperm counts have also been suggested as possibly associated with
phytoestrogens, but more recent work has not confirmed this pattern.28

Infants fed soy formula receive relatively high doses (per unit of body
weight) of phytoestrogens during a developmental stage at which permanent
changes are theoretically possible.29- 31
Delayed effects of soy-based infant formula on subsequent child or adult health
have thereby been postulated,30,31
generating substantial controversy in the lay and medical press. However,
effects on human pubertal and reproductive development of phytoestrogen exposure
in infancy have not been systematically investigated.13
Given the paucity of data on the long-term effects of soy formulas, their
widespread use, and expressed concerns about their safety,30,31
we undertook a study to examine the association between exposure to soy formula
in infancy and any subsequent possible effects on adult health, focusing on
outcomes that could be estrogen related.

Methods

Study Design

This was a retrospective cohort study of young adults, 20 to 34 years
of age, who as infants had participated in multiple controlled but nonrandomized
feeding studies conducted at the University of Iowa during the years 1965
to 1978. A telephone interview was conducted between March and August 1999
with eligible subjects who could be located and who agreed to be interviewed.
The study was approved by the University of Pennsylvania Committee on Studies
Involving Human Subjects.

The Cohort

Methods and procedures used in the original feeding studies have been
previously described.32,33 Briefly,
participants were healthy term infants with birth weights of more than 2500
g whose mothers elected not to breastfeed. With few exceptions, they were
white, reflecting the population in and around Iowa City. Infants were enrolled
before the age of 9 days and were studied through 16 weeks of age. Assignment
to study formulas was performed in rotation, in that all available infants
were assigned to whatever formula was being studied at the time. Once enrollment
of the predetermined number of infants was completed, all available infants
were assigned to the next study formula. Infant characteristics or parental
preferences were not taken into account. The same formula was fed for the
entire study period. Selected solid foods were received by some infants. The
study protocols involved periodic measurements of weight and length, measurement
of formula consumption, and determination of selected serum chemical indexes.
The actual phytoestrogen content of the infant formulas was not measured.

Criteria for inclusion in the present follow-up study were that the
formula fed be classifiable as "milk based" or "soy based," ie, containing
protein derived from cow milk or isolated soy protein or, in the case of one
formula (12 subjects in follow-up), from soy flour. Individuals who received
formulas with both soy and cow milk protein were excluded. In addition, subjects
had to have completed the original feeding study as planned (about 85% of
enrolled infants). Those who were adopted were excluded from follow-up. Also
excluded were individuals who were profoundly disabled who would not have
been able to be interviewed, deceased individuals, and individuals from countries
other than the United States, because of difficulty in tracking and differences
in lifestyle and language barriers.

Subject Follow-up

A search was conducted to locate the 952 subjects of the original cohort.
The search used a variety of approaches, including the use of national telephone
and address directories. Records of the parents' and often grandparents' names
and addresses were also available from the original study. Efforts to locate
subjects were aided by the fact that many of the individuals born from 1966
through 1971 had been located when they were 8 years old as part of a previous
follow-up study.34

Study Outcomes

Information on outcomes and a large number of potential confounding
factors was obtained by trained interviewers using a structured, standardized
telephone interview that took 30 to 60 minutes to complete. Reliability checks
were done on 5% of questionnaires. Outcome variables were selected for investigation
a priori based on 3 criteria. They were expected to be (1) potentially related
to estrogenic effects, (2) clinically important, and (3) likely to have a
sufficient sample size to detect a clinically relevant effect, based on a
priori standard type I and type II error levels. Because potential estrogenic
effects may be different in men and women, and many reproductive and sexual
outcomes are sex-specific, these outcomes were selected and analyzed separately
by sex.

The outcomes chosen for primary analysis in women were adult height,
usual weight since the age of 18 years, usual body mass index, pubertal maturation
(age at menarche, age when breasts developed enough to start wearing a bra),
number of days between periods (during times when not using birth control
pills, shots, or implants), number of days requiring pads or tampons, regularity
of menstrual period, menstrual flow, pain with menstrual period (none, mild,
severe), physical symptoms of pain (eg, dysmenorrhea, headaches), breast tenderness
during menstrual cycle, premenstrual symptoms, breast size (bra cup size),
reproductive outcomes (number of pregnancies, deliveries, abortions, and other
complications), and education level attained as a proxy measure for intelligence.

In men, in addition to adult height, usual weight, and education level,
outcomes investigated in the primary analyses included pubertal maturation
(age at first ejaculation, age when voice changed, age when hair began to
grow on chest, face, or pubic area) and pregnancy outcomes in sexual partners
impregnated by the male study subjects. Other outcomes, such as congenital
malformations in the offspring of study subjects, hormonal disorders, testicular
cancer in men, and homosexual orientation, were included as secondary outcomes
but were not expected to provide definitive results because these events were
expected to occur too infrequently.

Confirmation was sought in the medical records of past and current physicians
and hospitals of reports by the subjects of primary or secondary outcomes.

Data Analysis

The subjects were classified into exposure groups based on the original
infant formula consumed. Descriptive statistics were performed on all the
variables under investigation by sex and exposure group.

To compute the adjusted RR estimates, a single set of potential confounding
variables was used: birth weight (obtained from records available from the
infancy studies), current age, usual body mass, parents' usual weight (slim,
average, somewhat overweight, extremely overweight) and height, presence of
any hormone disorders (thyroid disease, treatment, use of steroids or growth
hormone), duration of cigarette smoking, average monthly alcohol consumption,
duration of use of soy foods as a major source of protein in diet (after infancy,
defined as "ever use" at least once per week), duration of vegetarian diet,
duration of dietary herbal supplement use, duration of use of mood-enhancing
drugs, average number of hours spent per week in strenuous sports or vigorous
work, average number of hours spent per day in sedentary activities, and sexually
transmitted diseases. For women, adjustment was made also for "ever use" of
birth control pills or progesterone injections or implants.

Distributions of continuous variables (such as body mass index, age
at menarche, age when voice changed) were characterized by means and SDs,
and differences between group means on these variables were initially compared
by the t test or Wilcoxon rank sum test, as appropriate.37 Hypotheses related to continuous outcomes were examined
using linear regression38 to estimate group
mean differences and 95% CIs while adjusting for the same list of potential
confounders.

All reproductive outcomes were evaluated, taking into account the correlation
among pregnancies for each woman.39 Live births,
miscarriages, and abortions were evaluated as a proportion of total number
of pregnancies. Full-term, preterm, and stillbirth outcomes were evaluated
as a proportion of total pregnancies, excluding miscarriages, abortions, and
ectopic pregnancies, since early losses in pregnancy were not eligible for
late pregnancy outcomes. Multiple births were evaluated as a proportion of
total live births. Unadjusted and adjusted RR estimates for all reproductive
outcomes were computed using log linear models with binomial error39 using PROC GENMOD in SAS statistical software, where
adjustments for multiple pregnancies (or correct denominator) were made by
specifying the appropriate offset for each subject. Adjustment was made for
the same list of potential confounders as for discrete variables plus the
presence of any reproductive organ disorder (polycystic ovarian syndrome,
endometriosis, blocked fallopian tubes, pelvic inflammatory disease, or other
problems with ovaries, uterus, or vagina), marital status, and education level
attained.

This study had 80% statistical power to detect clinically relevant differences
between the groups. For example, the study had sufficient power to detect
height differences of 0.9 and 0.8 in (2.3 and 2.1 cm) and weight differences
of 9.5 and 10.3 lb (4.3 and 4.6 kg) in men and women, respectively; a 1-day
difference in menstrual cycle length; and a difference of 0.45 years in age
at menarche. For dichotomous outcomes, we could detect statistically significant
relative risks (RRs) of 2.8 or greater for outcomes with an incidence of approximately
5%, 2.1 for outcomes with an incidence of 10%, or 1.5 for outcomes with an
incidence of 30%.

Results

Of the 952 subjects in the infant cohort, 48 were ineligible because
they had used formula with both soy and cow milk (n = 26), were from countries
other than the United States (n = 7), were adopted (n = 2), were disabled
(n = 3), or were deceased (n = 10), with causes of death accidental except
for 2 lymphoma/leukemia cases in the cow milk group. Of the 904 who were eligible,
811 were interviewed: 248 (87.9%) of 282 fed soy formula and 563 (90.5%) of
622 fed cow milk formula (Figure 1).
The groups differed in age (P = .001), with the soy
formula group having more individuals in the youngest and the oldest groups
compared with the cow milk formula group, whose age clustered in the 25- to
29-year range (Table 1). The only
other differences between the study groups were in use of asthma or allergy
drugs (soy greater than cow milk, P = .08 for men
but P = .047 for women) and a tendency for sedentary
activities (P = .77 for men but P = .05 for women).

No statistically significant differences were noted for either women
or men for adult height, usual weight, usual body mass index, or any of the
indexes of pubertal maturation (Table 2, Table 3, and Table 4).

Regarding pregnancy history, 54 (42.2%) of 128 women fed soy formula
as infants reported 1 or more pregnancies vs 128 (47.8%) of 268 women fed
cow milk formula as infants (P = .43). A total of
366 pregnancies (117 in the soy formula group and 249 in the cow milk formula
group) were reported by these 182 women. No differences were seen in pregnancy
outcomes (Table 3).

Evaluation of a large number of other outcomes selected a priori for
secondary analyses (eg, cancer, reproductive organ disorders, hormonal disorders,
libido dysfunction, sexual orientation, and birth defects in the offspring)
also did not show statistically significant differences in unadjusted analyses
between the 2 formula groups, in either women or men, although the sample
sizes for these analyses were too small to be definitive (data available from
the authors on request). Only regular use of weight control medications (ie,
daily, for at least 3 months) was borderline significantly higher in women
fed soy formula as infants (unadjusted RR, 1.70; 95% CI, 1.01-2.87).

A total of 228 subjects reported primary or secondary outcomes investigated
in this study. Almost two thirds (n = 146 [64.0%]) of these subjects granted
consent to review their medical records. After allowing for incomplete and/or
inaccurate physician addresses or unavailable records (n = 24) and for conditions
that could not be validated because they were self-diagnosed (n = 7), the
medical conditions of 115 subjects were potentially available for validation.
Records were actually obtained for 81 subjects, for a physician response rate
of 70.4% (81/115). The medical record review showed that of 106 medical conditions
reported, most were confirmed by the information recorded in the medical records
in both groups (33 [84.6%] of 39 in the soy formula group and 59 [88.1%] of
67 in the cow milk formula group).

Comment

Based on a 1998 infant feeding survey (Paul Harris, Ross Products Division,
Abbott Laboratories, Columbus, Ohio, written communication, April 2001), 18%
of infants are fed soy formula sometime during the first year of life. Given
the 2000 US Census estimate of nearly 4 million US infants younger than 1
year,40 an estimated 750 000 US infants
are fed these formulas each year. Even if the adverse outcomes under consideration
here were relatively uncommon, the potential for a major public health impact
is large. Conversely, insupportable allegations of adverse effects can affect
a large proportion of the population, denying them access to a useful type
of infant feeding product. Accurate assessment of any risk associated with
exposure to soy formula is important, and our study has yielded no systematic
cause for concern.

Our findings are consistent with other studies that found no changes
in weight and height or effects on puberty or fertility associated with the
consumption of soy isoflavones.10,11
However, we believe this to be the largest controlled study evaluating the
long-term effects of exposure to soy formula in infancy. This study had sufficient
statistical power (>80%) to detect clinically significant differences between
the groups in most outcomes, as is reflected in the relatively narrow CIs
presented. The study results were unequivocally negative across a large number
of outcomes that potentially may be influenced by the estrogenic or antiestrogenic
activity of phytoestrogens. This study found mostly neither positive nor negative
effects in subjects exposed in infancy to soy formula when compared with those
exposed to cow milk formula. From among the many different factors studied,
significant findings were seen only for slightly longer duration of monthly
menstruation and for greater discomfort with menstruation. The prolongation
of menstrual bleeding was small and was not accompanied by heavier bleeding.
Both findings were borderline positive and were 2 of many that were tested.
To place this in perspective, if we were to consider a Bonferroni adjustment
for the number of hypotheses investigated in this article, neither of these
2 findings would be considered even close to statistically significant at
the resulting stricter level of 0.05/30 = 0.0017.41
Furthermore, the clinical significance of these findings is not known.

Evidence in animals of reproductive disturbances associated with ingestion
of feed rich in estrogenic substances includes a lower conception rate in
sheep after prolonged grazing in clover pastures rich in isoflavones,17- 19 infertility in cattle
after consuming feed containing coumestrol,20
decreased fertility in captive cheetahs fed dietary estrogens,21
hyperestrogenism in pigs fed diets containing zearalenone,22
and uterotropic effects in mice fed soybean.16,23- 25
In contrast, a study of rhesus monkeys fed soy isolates for 6 months observed
no adverse effects on the reproductive systems of either sex, as evaluated
by reproductive hormone concentrations and organ weights at autopsy.42 Ours is the only epidemiologic study, to our knowledge,
that examines the possibility of infertility in young adults who were fed
soy formula in infancy. No statistically significant effects were seen on
fertility as measured by pregnancy or on miscarriage, medical abortion, or
ectopic pregnancy rates.

In a population-based cohort study in the United Kingdom,43
a vegetarian diet during pregnancy was associated with a 5-fold higher risk
of hypospadias, although regular consumption of soy products was not significantly
different between mothers with and without affected offspring. In our study,
rates of neither genital nor urologic birth defects in the offspring of subjects
fed soy formula in infancy were significantly different from those fed cow
milk formula.

Isoflavones from soy formulas are well absorbed by infants, as evidenced
by their presence in plasma44 and their urinary
excretion in amounts representing 13% to 38% of intakes. However, soy isoflavones
are largely conjugated. Unconjugated isoflavones are postulated to be extensively
metabolized by infants into glucuronide and sulfate conjugates that are likely
to exert low or negligible biological activity.45
In addition, it is unknown when an infant acquires the flora necessary to
metabolize isoflavones. Transit time, which is reduced in infants, may result
in less absorption.10 It may well be that phytoestrogens
are lightly bound to many of the proteins that bind estrogens and reduce their
activity. Alternatively, the newborn period could be a time when exposure
has little effect. This study does not provide data on the short-term activity
or effects of phytoestrogens. However, these results are reassuring regarding
the long-term effects of phytoestrogen exposure of this type.

Our study has several limitations. Isoflavone levels were not measured
in the soy formulas fed to the subjects. However, manufacturing processes
have changed very little since the study formulas were made. Isoflavone content
of the study formulas can, therefore, be assumed to have been similar to that
in currently marketed soy formulas, which has been reported to range from
32 to 47 mg/L.44 At the usual intake volumes
for infants during the first 16 weeks of age, isoflavone intake probably ranged
from 4.2 to 9.4 mg/kg per day. Soy flour has at least twice the isoflavone
content of soy protein isolate, so the isoflavone intake of the infants fed
the soy flour formula may have been between 9 and 16 mg/kg per day.

Selection bias in the way infants were assigned to study formulas was
unlikely. Although assignment to formula group was not randomized, it was
systematic in a fashion not likely to be related to outcome. By achieving
relatively complete ascertainment of the soy formula and cow milk formula
cohorts, we limited the potential of selection bias due to loss to follow-up
or participation refusals. Since phytoestrogens are found in many foods,46- 49 exposure
in infancy and adulthood to a variety of foods could mask any specific postulated
effects of soy formula. We controlled in multivariate analyses for total duration
of using soy products as a major source of dietary protein, use of herbal
supplements, relying on a vegetarian diet, and significant alcohol consumption.
No biological effects of infant soy exposure were detectable.

An important strength of this study is that the ascertainment of exposure
in infancy to soy formula or cow milk formula was not dependent on subject
or parental recall, because the information on exposure was obtained from
the research records from the initial clinical trials. In addition, we sought
the medical records of subjects with outcomes to validate the information
on medical outcomes obtained by interviews, with reassuring results.

Obtaining information on other exposure to soy during childhood and
adulthood, as well as information on other exposures and on the outcome variables
of interest, could be subject to recall bias because the respondents' recollections
could be flawed or intentionally distorted. Since the study hypotheses regarding
the outcomes were not known to the subjects and because the outcomes of interest
to the initial clinical trials were not the same as the outcomes of interest
to the follow-up cohort study, differential recall bias between the exposure
groups is unlikely. Nevertheless, nondifferential recall can bias the results
toward the null.

Interviewer bias should not be an issue in this study because we used
structured interviews and the interviewers were blinded to the study hypotheses
and to the formula group assignment of the subjects in the initial clinical
trials. Because of the wide-ranging contents of the interview, the interviewers
could not guess the association of interest to the study.

Misclassification bias50 could have occurred
in several ways in this study. Misclassification of initial exposure is unlikely,
since this information was objectively recorded in the research records of
the subjects during the initial clinical trials. Outcome misclassification
is likely to vary according to the specific outcome of interest, since respondents
are more likely to answer correctly when asked about the height or education
level attained than when asked about abortions or sexual preference. We attempted
to confirm some of this information by review of the current medical records.

Lack of generalizability of the conclusions might be a problem because
most subjects were from the Midwest, had higher socioeconomic status, and
were white. Although for reproductive outcomes it is difficult to imagine
that the results could not be generalized to other populations, it is possible
that for other outcomes, such as sexual preference and education level attained,
the results may be less generalizable to other ethnic or geographic groups.
On the other hand, the relative homogeneity of the cohort studied with respect
to socioeconomic status is useful in ensuring the validity of the comparisons
across the 2 exposure groups, since there is less risk of confounding by demographic
or socioeconomic status.

Finally, we cannot, of course, extrapolate from these findings on short-term
exposure to phytoestrogens in soy formulas the effects of long-term exposure
to phytoestrogens, nor to outcomes that we did not study, including longer-term
outcomes that would not have occurred yet.

In conclusion, for more than 30 primary hypotheses that were tested,
the observed differences between subjects exposed in infancy to soy formula
vs cow milk formula were small and few reached statistical significance. The
results with regard to menstruation should be interpreted with caution, given
that the clinical significance of slightly prolonged menstrual bleeding in
the absence of greater menstrual flow is not known. Given the large number
of comparisons evaluated in these analyses, the few marginally significant
findings may be due to chance. Although perhaps these few marginal positive
findings should be followed up in future studies, the findings of the current
study are reassuring about the safety of soy infant formula.

Adler JH, Trainin D. The apparent effect of alfalfa on the reproductive performance of dairy
cattle. In: Proceedings of the Fourth International Congress on Animal Reproduction
and Artificial Insemination; June 5-9, 1961; The Hague, the Netherlands.

Fomon SJ, Rogers RR, Ziegler EE, Nelson SE, Thomas LN. Indices of fatness and serum cholesterol at age eight years in relation
to feeding and growth during early infancy. Pediatr Res.1984;18:1233-1238.